The present invention relates generally to the field of cellular biology and, more particularly, to compounds, formulations, and methods for modulating autophagy to treat a wound or skin related disease or condition. The name, “autophagy,” also known as autophagocytosis, is derived from the Greek words meaning “eat” and “self.” Autophagy is generally defined as self-digestion.
Autophagy is primarily a lysosomal salvage or recycling pathway that is commonly used by cells to perform homeostatic functions by degrading aging proteins and organelles and reabsorbing nucleotides, amino acids, and free fatty acids for new molecule synthesis and ATP production. Autophagy may be up-regulated in response to extra- or intracellular stress and signals such as starvation, growth factor deprivation, ER stress, and pathogen infection as a cellular-level self-preservation mechanism. In some pathologic conditions, it may be desirable to stimulate the autophagy process, while in other pathologic conditions, such as wound healing, it may be desirable to suppress or slow the autophagy process to reduce the destruction of cells. Thus, modulation of autophagy may invoke either enhancing/stimulating or suppressing/slowing the cell growth and cell death process.
The most common form of autophagy involves (1) the formation of an isolation membrane, which extends and the termini ultimately (2) fuse to encompass the cellular contents within a double-membrane vesicle known as an autophagosome. The autophagosome then (3) fuses with a lysosome that provides enzymes to (4) digest the contents of the autophagosome, which contents then become available to the cell again as raw materials or building block nutrients. Autophagy exhibits some similarities to the parallel proteasome degradation of ubiquitin-tagged proteins, but differs in that the autophagosome contains not only proteins, but also cytoplasm, mitochondria, organelles and other cellular structures. In this sense it is known as a bulk degradation system.
Since the process of autophagy can be both beneficial and detrimental to the cell depending on external factors and conditions, the process must be tightly regulated. Both yeast and mammalian systems have been studied and utilize up to 36 proteins. FIG. 1 illustrates the process in mammals.
The yeast autophagy-related gene product (Atg8) has three mammalian homologues: (1) LC3, (2) GABAA receptor-associated protein (GABARAP), and (3) Golgi-associated ATPase enhancer (GATE-16). Among them, LC3 is most actively studied and frequently used as a mammalian autophagy marker. Shortly after translation (proLC3), LC3 is processed at the C-terminus by Atg4A or Atg4B into LC3-I. Upon induction or enhancement of autophagy, LC3-I is conjugated to the substrate phosphatidylethanolamine (PE) via E1 (yeast Atg7) and E2 (yeast Atg3). The PE-LC3-I conjugate is referred to as LC3-II. This conjugation occurs in the process at the point of autophagosome formation and leads to the conversion of soluble LC3-I to the autophagic vesicle associated LC3-II. This lipid conjugation allows LC3-II to be used as a marker of autophagy activity.
Wound progression is caused by many mechanisms including local tissue hypo-perfusion, prolonged inflammation, free radical damage, apoptosis, and necrosis. These are typically broken up into three stages following the injury including; (i) the inflammation phase, (ii) cell proliferation phase and (iii) remodeling phase. Each one of these phases is linked to a biologically and histologically unique fingerprint and autophagy plays a special role at each phase. For example, during the inflammation stage, autophagy is initially protective of tissue in that it attempts to keep the wound edge cells from dying. In the proliferative stage, during which involves cell multiplication and migration of multiple cell types to close the wound, autophagy helps to regulate β1-integrins and other cell migration proteins to form a controlled line of cells at the leading edge of the wound. In remodeling, the direction of these migrating cells is often controlled by the directionality and deposition of collagen that is secreted by these transformed fibroblasts as pro-collagen. Collagen along with other components such as fibronectin and lamin help regenerate the basement membrane during the proliferation and migration stages.
During the wound healing process collagen fibrils provide the structural topology, rigidity and organization that allows for proper cell migration. In chronic wounds and burns this environment is subjected to a variety of factors including an altered pH, chronic inflammation and often a loss in the basement membrane including the essential collagen tracts that are used by migrating wound cells (e.g. keratinocytes, fibroblasts, myofibroblasts) to fill the wound and restore a layer of intact skin to protect the body from invading microbes and environmental factors (e.g. temperature regulation). During the natural healing process skin cells secrete the soluble form of collagen known as pro-collagen that can form higher order structures enzymatically or undergo an entropy driven self-assembly process (Prockop, D. and D. Hulmes (1994). Pro-collagen is secreted from the cell as a triple helix, which can self-assemble into fibrils due to its liquid crystal nature. This process is highly dependent on the pH, ionic strength, temperature, and concentration. Fibrils then are formed into individual collagen fibers, which are randomly assembled into higher order 3-dimensonal networks and structures.
U.S. Pat. Nos. 6,599,945 and 7,094,809 disclose several hydroxytolan compounds and their use in inhibiting the formation of infectious herpes virus particles or for treating gonorrhea caused by Neisseria gonorrhoeae. WO2009/126700 discloses the use of similar compounds for skin care, such as UV radiation, and cosmetic uses. And U.S. Pat. No. 8,716,355 (WO2011/0130468) and U.S. Pat. No. 8,680,142 (WO2011/0160301) disclose similar hydroxytolans for use as anti-tumor agents. However, the potential utility of these, or any other hydroxytolans as autophagy-modulating compounds was unknown until the making of the present invention. U.S. Pat. Nos. 6,008,260, 6,197,834 and 6,355,692 disclose certain hydroxylated stilbenes, and specifically resveratrol. None of these references disclose the use of such modified tolan or stilbene compounds as autophagy modulating agents.
It would be advantageous if the process of autophagy could be modulated, that is stimulated or enhanced in some conditions, and slowed or suppressed in other conditions.